In traumatic and ischemic brain injury, both axons and dendrites are highly vulnerable neuronal structures during glutamate excitotoxicity. Both structures are enriched with microtubules that have important subcellular transportation functions. The stability of microtubules is mainly controlled by the presence of microtubule-associated proteins, including the axonally-enriched tau and the dendritically-enriched MAP2a/2b (MAP2). Here, we hypothesize that both tau and MAP2 are highly susceptible to combined attacks by the cell death-linked cysteine proteases calpain and caspase-3. The degradation of tau and MAP2 likely leads to the depolymerization of microtubules and significant structural and functional compromise of axons and dendrites. We have unpublished and novel preliminary data showing that both Tau and MAP2 are equally vulnerable to in vitro calpain and caspase-3 proteolysis. In SPECIFIC AIM 1 of the current proposal, we will first identify major calpain and caspase-3 cleavage sites of Tau and MAP2, respectively and we will use that knowledge to raise fragment-specific antibodies. Then in SPECIFIC AIM 2, we will examine proteolytic vulnerability of both Tau and MAP2 in cultured rat cerebrocortical neurons subjected to excitotoxic challenge, and to calpain-dominant maitotoxin challenge and caspase-dominant EDTA challenge (positive control models). By comparing the Tau and MAP2 fragmentation patterns in excitotoxic injury to those generated in these positive controls, we will assess the relative contributions of calpain versus caspase-3 to these proteolytic events. We will also use these cell culture paradigms to validate that the Tau and MAP2 fragment-specific antibody tools we developed in SPECIFIC AIM 1 in fact could detect calpain and capase-3 proteolysis of the respective proteins in situ. In SPECIFIC AIM 3, we will extend our study to an in vivo rat model of traumatic brain injury (controlled cortical impact). We will examine the relative contribution and temporal profile of the dual calpain and caspase-3 attack of axonally enriched Tau and dendritically enriched MAP2. Lastly, we will test the novel hypothesis that the combined use of potent calpain and caspase inhibitors (when given in appropriate timeframes) would result in synergistic and maximal protection of Tau and MAP2 proteins against proteolytic degradation.